The adult nervous system can be thought of as a network of neuron cell bodies connected to each other by thin processes called axons. During development, these axonal extensions are guided to their target by patterned chemical cues in their environment. Failure to reestablish these axonal connections is a major cause of the persistent disabilities of a number of disease conditions, including: spinal cord injury, traumatic brain injury and stroke. The leading tip of an axon, known as a growth cone, uses cell surface receptors to recognize these chemical cues. The growth cone is also a mechanical structure that pulls on its surroundings to move forward. In theory, cues could have an indirect role in influencing the coupling of the growth cone's locomotive machinery to its surroundings. However, I have recently shown that at least one of these cues - netrin-1 - is directly used for traction. In other words, the ability of netrin-1 to attract the growth cone reflects its ability support the growth cone's mechanical pulling. My current work examines a protein called focal adhesion kinase (FAK) within the growth cone that links this cue to the cytoskeleton. I have discovered that the mechanical tension felt on this protein activates its catalytic activity resulting in biochemical cascades that reinforces the link to the cue. In this application, I propose to use optical laser tweezers, magnetic tweezers, super resolution imaging techniques and nano-fabricated pillar arrays to address four other fundamentally important lines of investigation that have emerged from my previous work: (1) Determine how FAK is attached to cytoskeleton and whether it is physical stretched in cells. (2) Test the effect of chemical composition and rigidity of the environment, as well as, changes between different neuronal populations on the pulling strength of the growth cone. (3) Examine whether other attractive axon guidance cues used for traction. (4) Explore whether mechanical tension felt on the cue and its receptor alters their function by inducing conformational changes. Michael Sheetz'lab offers a unique environment to learn and utilize the techniques necessary to address these questions. Two additional co-mentors will provide complimentary expertise: John Hunt is world leader on protein purification and structure, while James Hone is an expert on generating cutting-edge nanofabricated devices. Insights gained from this project will further our basic understanding of axon guidance and therefore contribute to the development of better regenerative strategies following injury of the nervous system. PUBLIC HEALTH RELEVANCE: Insights gained from this project will further our basic understanding of axon guidance. Failure to reestablish axonal connections is a major cause of the disabilities seen following a number of disease conditions, including: spinal cord injury, traumatic brain injury and stroke. This project therefore contributes to the development of better regenerative strategies following injury to the nervous system.